Self-renewal and pluripotency are defining characteristics of stem cells common in both embryonic stem (ES) cells (derived from developing embryos) and induced pluripotent stem (iPS) cells (derived from differentiated somatic cells). These cells can self-renew for a prolonged time in vitro and retain differentiation potential (pluripotency) to give rise to all possible cell types of developing organisms. Due to such exceptional characteristics, ES and iPS cells have been studied extensively for understanding the molecular basis of early development, and serve as useful instruments in drug discovery and establishing human disease models. Since transcription factors, Oct4, Sox2, and Nanog have been identified in ES cells as playing master regulatory roles in self-renewal processes, advances in high-throughput technologies have led to the discovery of additional cofactors of master regulators. These discoveries have allowed us to better understand the regulatory mechanisms of stem cell self-renewal. On the other hand, our understanding of the regulatory mechanisms that retain the differentiation potential of stem cells is significantly limied and such mechanisms have not yet been systematically examined. Obviously, complete understanding of both self-renewal and pluripotency would be crucial in harnessing the full potential of pluripotent stem cells in future therapeutic applications. The objective of the proposed research is to better understand the regulatory mechanisms governing the differentiation potential of ES cells. Our primary questions are 1) What are the transcription factors involved in maintaining and controlling the differentiation potential of pluripotent stem cells? 2) How do they influence the direction of early lineage specification? 3) What is the nature of the regulatory networks they form, and how do these networks intertwine with the previously known self-renewal network? We will address these questions systematically by applying a signature based screening method in combination with various systems biology tools. Successful completion of this project will not only allow us to functionally characterize novel regulators controlling the differentiation potential of stem cells, but also illustrate the full viw of regulatory mechanisms governing the hallmarks of pluripotent stem cells. Furthermore, outcomes of this proposal will provide a foundation for manipulation of stem cells to control cell fates towards desired lineages, and contribute to the advances in stem cell based cell therapies.